Each year in the United States alcohol abuse leads to more than 100,000 deaths and results in economic costs of more than 180 billion dollars;it is further estimated that almost half of all patients in hospitals have health problems exacerbated by alcohol use. Such tremendous costs have motivated intensive research efforts to understand how this drug affects brain function. Yet despite these efforts, there is no consensus as to how ethanol acts at a neuronal level. A prevailing hypothesis has been that ethanol enhances the function of GABAA receptor-mediated signaling in brain but whether specific GABAA receptor subtypes are involved, and if so how ethanol enhances their function are unanswered questions. Our prior work has suggested that d subunit-containing, extrasynaptic isoforms of GABAA receptors are important targets for the low concentrations of ethanol achieved during moderate social consumption. In a collaborative effort, we propose to systematically examine key factors that influence the sensitivity of recombinant and native versions of extrasynaptic GABAA receptors. The grant is organized into three aims. Aim 1 proposes to make use of a novel "functional tag", a single amino acid substitution in the d subunit, which renders it sensitive to benzodiazepines. The tag will allow us to carefully correlate the presence or absence of this subunit with ethanol sensitivity. Aim 2 utilizes laser photolysis of chemically caged GABA to map sensitivity to ethanol and to other GABAA receptor modulators on cerebellar neurons. Aim 3 tests the hypothesis that d subunit incorporation endows both recombinant and native GABAA receptors with nanomolar sensitivity to the sedative hypnotic drug Gaboxadol/THIP. Such discriminative pharmacology would explain the powerful behavioral actions of THIP as a sedative and would validate the compound as a tool for selectively activating d subunit containing GABAA receptors. The knowledge obtained from these experiments will give us a more thorough understanding of how alcohol and sedative hypnotic drugs affects brain function. In particular, the results should define the contributions of a key ethanol target to acute alcohol intoxication. Molecular level identification of ethanol targets is a prerequisite for the development of rational therapies to treat alcohol-related cognitive impairment and alcohol addiction. PUBLIC HEALTH RELEVANCE: We propose to study the molecular details of ethanol action at extrasynaptic GABAA receptors, a subset of inhibitory neurotransmitter receptors implicated as key targets in both acute alcohol action and in adaptation to chronic alcohol exposure. Validation of this exciting new class of ethanol targets and identification of the determinants of ethanol action will enable the development of new therapies to address alcohol addiction and the chronic changes in brain function that occur during alcoholism.